Mar 11, 2026

Public workspaceA TIRF microscopy-based assay to monitor LRRK2-dependent endo-lysosome exocytosis

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld4nj8l5b/v1
  • Ayan Adhikari1,
  • Sreeja V Nair1,
  • Suzanne Pfeffer1
  • 1Stanford University School of Medicine and Aligning Science Across Parkinson's
  • Team Alessi
Suzanne R Pfeffer
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld4nj8l5b/v1
Protocol Citation: Ayan Adhikari, Sreeja V Nair, Suzanne Pfeffer 2026. A TIRF microscopy-based assay to monitor LRRK2-dependent endo-lysosome exocytosis. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4nj8l5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 11, 2026
Last Modified: March 11, 2026
Protocol Integer ID: 313111
Keywords: Lysosome exocytosis, LRRK2 kinase, TIRF microscopy, lysosome exocytosis event, lysosome exocytosi, lysosome exocytosis in htert, acidic compartments such as lysosome, lysosome, fluorescence emission of phluorin, rpe cell, fluorescence emission, tirf microscopy, total internal reflection fluorescence, using total internal reflection fluorescence, microscopy, plasma membrane, phluorin, based assay, acidic compartment, monitoring cd63, specific protein depletion, sudden shift in ph, sirna transfection
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
Abstract
Here we describe a method for monitoring CD63+ endo-lysosome exocytosis in hTERT-RPE cells stably expressing CD63-pHluorin. The fluorescence emission of pHluorin, a pH‑sensitive fluorescent probe, is quenched within acidic compartments such as lysosomes and multi-vesicular bodies (Miesenböck et al., 1998; Verweij et al., 2018). When these compartments fuse with the plasma membrane, the sudden shift in pH from acidic to neutral causes the probe to fluoresce. CD63-pHluorin enables monitoring of fusion events at the plasma membrane using Total internal reflection fluorescence (TIRF) microscopy. Powerful ExoJ software (Liu et al., 2024) enables automated quantitation of individual events. This protocol includes siRNA transfection to monitor the effects of specific protein depletion on endo-lysosome exocytosis events.
Materials
  1. DMEM high glucose (Cytiva, SH30243.02)
  2. Penicillin/Streptomycin (Cytiva, SV30010)
  3. Rat tail collagen (Gibco, A1048301)
  4. Opti-MEM, Reduced serum medium (Gibco, 31985070)
  5. DharmaFECT 4 Transfection Reagent (Horizon Discovery, T-2004-02)
  6. siGLO Red Transfection Indicator (Horizon Discovery, D-001630-02-05)
  7. µ-Slide 8 Well (ibidi, 80807)
  8. Leibovitz's L-15 Medium, no phenol red (Gibco, 21083027)
  9. hTERT-RPE-CD63-pHluorin cells
  10. pLenti-pHluorin_M153R-CD63 (Addgene #172117)
  11. pCMV-lyso-pHluorin (Addgene #70113)
  12. Ionomycin calcium salt (Sigma-Aldrich, I3909)
  13. MLi-2 (MRC PPU Reagents and Services, U. Dundee, #1627091-47-7)
  14. Nikon Ti-E inverted microscope with Andor iXon+ EMCCD camera (Model- DU885)
Troubleshooting
Plate Cells
Cells are imaged in glass bottom chambers and plated sparsely on chamber slides coated with rat tail collagen, at least 16 hours before imaging. Control reactions should be treated with Calcium ionophore, ionomycin (750nM) for maximum secretion (exocytosis must be visualized starting upon drug addition from t=0 minutes) or the LRRK2 kinase inhibitor, MLi-2 (250nM) added for 60 minutes before imaging.
Optional siRNA transfection in hTERT-RPE cells
OPTIONAL: To evaluate the role of various proteins in exocytosis, cells can be transfected with siRNAs to decrease specific target proteins. Here, siGLO red transfection control is co-transfected to enable identification of cells that received the siRNA, for further analysis.
Plate 1.5 × 10⁵ hTERT-RPE cells per well of a 6-well plate in complete medium (DMEM + 10% FBS + 1% penicillin/streptomycin).
Incubate the cells for 16–18 h to allow them to attach and recover.
Prepare the transfection mix as follows (per well of a 6-well plate):

Transfection mix recipe

Add the indicated reagents to Tube 1 and Tube 2, and bring each up to 200 µL with Opti-MEM. Mix gently by pipetting up and down 3–4 times
Incubate both tubes at room temperature for 5 min.
Add the contents of Tube 1 to Tube 2, and mix gently by pipetting up and down 3–4 times.
Incubate the siRNA–DharmaFECT complexes at room temperature for 20 min.
Aspirate the old medium from the cells and add 1.6 mL of fresh complete medium [DMEM + 10% FBS + 1% penicillin/streptomycin] per well (6-well plate).
Add the 400 µL transfection mixture dropwise to the cells and gently swirl to distribute evenly.
Incubate the cells for up to 72 h.


Preparation of TIRF imaging glass chamber slides
Coat each well of an 8-well chamber slide with 50 µg/mL rat tail collagen for 45–60 min at room temperature.
Aspirate and Aspirate rinse each well three times with sterile water.
Rinse twice with complete medium and the chamber is now ready for cell plating.
At 48 h post-transfection, trypsinize the cells and plate onto the collagen-coated 8-well chamber at low confluency such that individual cells can be identified during TIRF imaging.
Allow cells to attach and recover (16-24 h) before imaging.
TIRF imaging
Aspirate the culture medium from each well of the 8-well chamber.
Gently rinse once with Leibovitz's L-15 Medium, no phenol red, supplemented with 10% FBS and 1% penicillin–streptomycin.
Add 0.3 mL of supplemented L-15 medium to each well.
Proceed to TIRF imaging.
TIRF operation protocol: Nikon Ti-E inverted microscope
This section is tailored to this specific microscope (Nikon Ti-E inverted with APO TIRF 100X, 1.49NA objective) adapted for TIRF microscopy.
Switch on the temperature controller (In Vivo Scientific, red switch) and turn the blower knob to maximum. Wait until the stage reaches 37°C.
On the 16-bit digital controller (Allied Scientific Instrumentation) switch on the shutters: AOTF, LMM5, and power: SCOPE (all red switches). Also switch on the desired laser line(s). Lambda XL (Sutter Instruments) is used as the light source and the wavelengths are selected using a quad pass dichroic mirror (Chroma 89000). NOTE: Use Laser 491nm for pHluorin and 561nm for sciGLO Red transfection control.
Once you switch on the laser, the main laser indicator will start blinking; when it stabilizes, turn the laser power ON. The indicator will change from green to orange.
Launch the Micro-Manager software (version 1.4; Edelstein et al., 2010). Load the configuration file that specifies microscope operations.
In the software, set the laser power as desired (for 491 nm laser we use 70% and for 561 nm the laser is set at 50% for siGLO). To achieve a 200nm (shallow) sample illumination distance, set the TIRF position to approximately 46970 (between 46900 and 47000) and set the camera gain initially to 10.
Place the slide on the appropriate adapter and mount it on the microscope stage. Use the 100X Apo TIRF oil-immersion objective.
In the software, select “Bright Field.” A yellow light beam will illuminate the sample on the microscope.
Use the focus knob to bring the sample into focus; sharp images are usually obtained between 4900 and 5200µm on the Z readout.
Turn off Bright Field illumination in the software and turn on the 491 nm fluorescence channel. An excitation beam will illuminate the sample. Increase the camera gain to 50 and fine-tune the focus on the screen. Once the focus is optimized, switch on the Perfect Focus System (PFS) and on the microscope body, press the “Memory” button located just above the display next to the PFS button. You do not need to turn PFS off when moving to another field, but minor focus adjustments can be made using the PFS controller; after any focus change, press “Memory” again to store the new position.
In the software, open the “Multiple Acquire” (Multi-D acquisition) window. Set the acquisition parameters: for example, acquire 400 frames with a 300 ms exposure (or interval) per frame. At the bottom of the window, specify the “SAVE” directory and base filename for your image sequence. File saving settings should always be ‘image stack file’.
Start image acquisition. Monitor the focus (PFS) as needed during the time-lapse (usually 2 minutes).
Image analysis
We generally record 2 minutes of video for further analysis.
Perform image analysis using the ExoJ plugin in Fiji/ImageJ as described in Liu et al. (2024)
https://doi.org/10.1242/jcs.261938
Open FIJI (Schindelin et al. (2012)) and drag the video file into the software. From the Plugins menu in FIJI, select ExoJ. A window titled “ExoJ: List of all available files” will appear, displaying your file name; select the file and click “Open.”

Figure 1. Launching ExoJ plugin in FIJI
Two windows will open: one displaying your video (file name starting with “DUP_…”) and another showing the analysis settings (“ExoJ: Event Detection”). Using the drawing tools, outline the cell of interest and define the region of interest (ROI), then add the ROI to the ROI Manager in FIJI.
In the Event Detection window, enter the analysis parameters (we typically use the same settings as reported in Liu et al. (2024) for quantifying exocytosis events), and click “Run.” The software will begin detecting exocytosis events; this step may take some time depending on the performance of the computer. When detection is complete, click “Next.”
Figure 2. Image showing parameters for vesicle detection
Hit "RUN" and then this window appears:

Figure 3. After hitting "RUN"....
You can verify individual exocytosis events by selecting a specific event in the "Detected Exocytosis List" window; the corresponding event will be highlighted in the "Secretion Preview" window. To add a new fusion event, use the rectangle selection tool in FIJI, draw a rectangle around the bright spot in the secretion preview and click on “Add”.
For further validation, use the “Spatial Dynamics” option in the "Detected Exocytosis List" window. Select an event and click “Spatial Dynamics” to generate a plot of intensity versus frame number. A valid exocytosis event should show a clear increase in intensity followed by a decrease over a defined range of frames.
Figure 5. Intensity profile of a valid exocytosis event
If the event does not follow the above criteria, as shown below, select the particular event in “Detected exocytosis list” and click “Remove”.
Figure 6. Intensity profile of an invalid exocytosis event
ESSENTIAL CONTROL REACTIONS
Control reactions should be treated with 750nM ionomycin for maximum secretion (exocytosis visualized starting from t=0 minutes) or MLi-2 (250nM) added for 60 minutes before imaging. Cells are maximally useful for 7-8 passages and should yield 20 events per two minutes in control conditions, ~2-5 events/2 min with MLi-2 and up to 60-75 events/2 minutes with ionomycin.

Figure 7. Example data showing LRRK2 kinase dependence







Protocol references

Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. (2010) Computer control of microscopes using μ Manager. Current Protocols in Molecular Biology. New York, Green Publishing Associates and Wiley Interscience. doi: 10.1002/0471142727.mb1420s92.
Liu, J., Verweij, F. J., van Niel, G., Galli, T., Danglot, L., & Bun, P. (2024). ExoJ–a Fiji/ImageJ2 plugin for automated spatiotemporal detection and analysis of exocytosis. Journal of Cell Science, 137(20), jcs261938.
Miesenböck, Gero, Dino A. De Angelis, and James E. Rothman. "Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins." Nature 394.6689 (1998): 192-195.
Schindelin, J., Arganda-Carreras, I.,Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ... & Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature methods, 9(7), 676-682.
Verweij, F. J., Bebelman, M. P., Jimenez, C. R., Garcia-Vallejo, J. J., Janssen, H., Neefjes, J., ... & Pegtel, D. M. (2018). Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. Journal of Cell Biology, 217(3), 1129-1142.